BACKGROUND: The ongoing opioid epidemic is driven in part by legitimate use of opioids prescribed for neuropathic pain. Targeting alternative pathways for pain control using non-addicting drugs is a major goal of neuropathic pain research. After peripheral nerve injury (PNI), cytokines and chemokines are upregulated centrally, where they contribute mechanistically to the pathogenesis of neuropathic pain. Reactive astrocytes in the dorsal horn exhibit a chronically activated, pro-inflammatory secretory (CAPS) phenotype characterized by secretion of numerous factors, including interleukin-6 (IL-6), chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 1 (CXCL1) and nerve growth factor (NGF). The post-PNI astrocytic CAPS phenotype contributes to both inflammation and neuronal hyperactivation via neuronal chemokine receptors, leading to neuropathic pain. Canonical NF-?B signaling is known to play a crucial role, but surprisingly, despite its documented importance in numerous inflammatory conditions involving most organs including the brain, non- canonical NF-?B signaling via the p52:RelB heterodimer has not been identified previously in PNI. New work from our laboratory demonstrates the novel finding that non-canonical NF-?B signaling by the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) / fibroblast growth factor-inducible 14 (Fn14) axis is prominent after PNI, especially in dorsal horn astrocytes, and may be responsible for transcriptional expression of Sur1- Trpm4 in these cells. Moreover, our preliminary data suggest that glial Sur1-Trpm4 plays a crucial role in regulating the expression of IL-6, CCL2 and CXCL1. Our central hypothesis is that, in dorsal horn astrocytes post-PNI, TWEAK-induced non-canonical NF-?B signaling is an upstream regulator of Sur1-Trpm4 expression, and that Sur1-Trpm4, in turn, regulates the expression of the downstream effectors, IL-6, CCL2 and CXCL1, which promote chronic neuroinflammation, neuronal hyperactivation and neuropathic pain. DESCRIPTION: This project has three mechanistic aims. In Aim 1, genetic (Tweak?/?, Fn14?/?, p100?/?, Abcc8?/?, either global or GFAP-specific or GFAP-&-site-specific) and pharmacological (anti-TWEAK antibody, glibenclamide) experiments will be carried out to determine the role of non-canonical NF-?B and Sur1 in GFAP-expressing glia in sciatic n. vs. dorsal horn in two neuropathic pain models: sciatic n. cuffing and sciatic n. exposure to HIV/gp120 protein. In Aim 2, we will expand upon in vivo and in vitro data from chromatin immunoprecipitation to establish the role of non-canonical NF-?B in the expression of functional Sur1-Trpm4 channels. In Aim 3, we will corroborate and expand upon in vivo and in vitro data to establish the role of Sur1- Trpm4 in regulating Ca2+/calcineurin-dependent transcription of IL-6, CCL2 and CXCL1. This project is the first to study non-canonical NF-?B in dorsal horn vs. sciatic n. glia in neuropathic pain, and will help identify novel druggable targets to ameliorate neuropathic pain using non-addictive drugs.